Fragments of leptin (ob protein)

ABSTRACT

A leptin or ob peptide or a functional derivative, analogue or variant thereof, which modulates body weight substantially by means of modulating energy utilization, a pharmaceutical composition containing such a compound, a process for the preparation of such a compound and the use of such a compound in medicine.

[0001] The invention relates to novel compounds, in particular to novelpeptides, to compositions containing such compounds and to the use ofsuch compounds in medicine.

[0002] The mechanism of the physiological regulation of energy balancein the body—food intake verses energy output—has been the subject ofdebate for many years. In a recent publication in Nature Y. Zhang et al,(Nature, 372, 425-431, 1994) suggest that one of the molecules whichplays a key role in energy balance regulation is the ob protein. Zhanget al also report the cloning and sequencing of both mouse and human obgene protein or leptin.

[0003] The structure of human leptin or (human ob protein) and its usein the modulation of body weight in animals is disclosed in UnitedKingdom Patent application Publication Number GB2292382. Thisapplication also discloses certain fragments of leptin which are alsostated to be capable of modulating body weight.

[0004] Collins et al in Nature, Vol 380, page 677, 1996 disclose thatthe weight reducing properties of leptin may be accounted for by anenhancement of energy utilization as well as decreasing food uptake

[0005] We have now discovered certain novel fragments of leptin whichsurprisingly are indicated to modulate body weight substantially bymeans of enhancing energy utilization. These fragments are thereforeconsidered to be of particular use in the treatment of nutritional andmetabolic disorders, particularly obesity and diabetes.

[0006] Accordingly, in a first aspect, the present invention provides apeptide or a functional derivative, analogue or variant thereof, whichmodulates body weight, substantially be means of modulating energyutilisation.

[0007] Preferably the modulation of body weight is a reduction of bodyweight.

[0008] Preferably the modulation of energy utilisation is via anenhancement of energy utilization.

[0009] Preferably, the peptide is a fragment of an ob protein,especially human ob protein, or a functional derivative, analogue orvariant thereof.

[0010] Hereinafter protein fragments (or peptides) will be referred towith reference to the amino acid sequence of human ob protein, using ananalogous abbreviation to the following: ‘the protein fragmentconsisting of amino acid residues 1 to 6’ is abbreviated to ‘ob1-6’.

[0011] Particular peptides include ob21-26 (MVPIQK), ob27 -32 (VQDDTK),ob33-36 (TLIK) , ob37-41 (TIVTR), ob42-54 (INDISHTQSVSSK), ob55 -56(QK), ob57-74 (VTGLDFIPGLHPILTLSK), ob93-105 (NVIQISNDLENLR), ob106-115(DLLHVLAFSK), ob116-149 (SCHLPWASGLETLDSLGGVLEASGYSTEVVALSR) andob150-167 (LQGSLQDMLWQLDLSPGC) especially ob57 -74 (VTGLDFIPGLHPILTLSK)

[0012] Suitably, the invention includes a peptide formed from any one ormore of the aforementioned particular peptides.

[0013] Favourably, the invention includes a peptide formed from any oftwo contiguous members of the aforementioned particular peptides.

[0014] As stated, the invention also extends to the functionalderivatives, analogues and variants of the peptides mentioned herein:

[0015] Functional derivatives includes salts and solvates of thepeptides mentioned herein and also the peptides of the inventionchemically modified by the attachment of groups or moieties so as toimprove the physical properties, such as stability, or the therapeuticproperties, for example the pharmacokinetic properties, of the protein.

[0016] Functional analogues includes functionally analogous peptideswherein one or more amino acids of the peptides mentioned herein arereplaced with alternative amino acids.

[0017] Alternative amino acids includes amino acids of alternativestereochemistry to the amino acids in ob protein.

[0018] Functional analogues also include small molecule agonists orantagonists of the peptides mentioned herein. Such compounds may beprepared and tested according to known procedures, for example thosedisclosed in GB2292382.

[0019] Salts include pharmaceutically acceptable salts, especiallypharmaceutically acceptable acid addition salts.

[0020] Acid addition salts of the peptides are prepared in a standardmanner in a suitable solvent from the parent compound and an excess ofan acid, such as hydrochloric, hydrobromic, sulphuric, phosphoric,acetic, maleic, succinic, or methanesulphonic. The acetate salt form isespecially useful. Certain of the compounds form inner salts orzwitterions which may be acceptable. Cationic salts are prepared bytreating the parent compound with an excess of an alkaline reagent, suchas a hydroxide, carbonate or alkoxide containing the appropriate cation.Cations such as Na⁺, K⁺, Ca²⁺ and NH₄ ⁺ are examples of cations presentin pharmaceutically acceptable salts.

[0021] Solvates include pharmaceutically acceptable solvates, such ashydrates.

[0022] It will be appreciated that the invention includes both peptideand non-peptide compounds.

[0023] In addition the invention includes sub-fragments of theparticular peptides ob21-26, ob27-32, ob33 -36, ob37-41 , ob42 -54,ob55-56, ob57-74, ob93-105, ob106-115,ob116-149 and ob150-167,especially ob57-74;or a peptide formed from any one or more, especiallyof any two contiguous members, of the said particular peptides; or afunctional derivative, analogue or variant thereof.

[0024] Suitable peptides or sub-fragments comprise at least 4 aminoacids.

[0025] The peptides of the invention are suitably prepared by usingconventional digestion methods, synthetic techniques or by use ofstandard expression methodology.

[0026] Thus in a further aspect, the present invention provides aprocess for the preparation of a peptide, or a functional derivativethereof, the process comprising the steps of:

[0027] hydrolysing the peptide, especially an ob protein and inparticular a human ob protein, into at least two peptide fragments;

[0028] separating the peptide fragments; and optionally thereafterpreparing a functional derivative thereof.

[0029] The hydrolysis of the protein is suitably effected by enzymicdigestion, using for example trypsin.

[0030] The separation of the required peptide is convenientlyaccomplished by use of an appropriate chromatographic means, such ascolumn chromatography.

[0031] The specific reaction conditions for the treatment of the obprotein, providing they are commensurate with the stability of therequired product, are determined by the nature of the particular reagentused, for examples when trypsin is the reagent then the reaction isnormally carried out within a temperature range of 25-40° C. and a pHrange of 7-9, preferably at 37° C. and pH 7.4.

[0032] As stated, the peptides of the present invention may also beprepared by conventional synthetic procedures, for example by use ofliquid or solid-phase peptide synthesis.

[0033] Accordingly, the present invention provides a synthetic peptideor a functional derivative, analogue or variant thereof, which modulatesbody weight, substantially be means of modulating energy utilisation.

[0034] Any of the peptides mentioned herein form part of the inventionas synthetic peptides.

[0035] Peptide bonded units of the proteins associated with the presentinvention can be prepared by standard peptide synthesis techniques usinga peptide synthesiser (Atherton, E. and Sheppard, R. C. (eds.) (1989)Solid Phase Peptide Synthesis: A practical approach, LRL Press, Oxford)followed by procedures appropriate to direct disulphide or amide bondformation.

[0036] Methods of well-known peptide synthesis are set forth by Ali et.al., J. Med. Chem., 29:984 (1986) and J. Med. Chem., 30:2291 (1987) andare incorporated by reference herein. Preferably, the peptides areprepared by the solid phase technique of Merrifield (J. Am. Chem. Soc.,85:2149 (1964)). However, a combination of solid phase and solutionsynthesis may be used, as in a convergent synthesis in which di-, tri,tetra-, or penta-peptide fragments may be prepared by solid phasesynthesis and either coupled or further modified by solution synthesis.

[0037] During synthesis, the side chain functional groups (e.g., —NH₂,—COOH, —OH, —SH) are protected during the coupling reactions. Normally,the α-amino group is temporarily protected as fluorenylmethoxycarbonyl(Fmoc) but other acid- or base-labile protecting groups can be used,e.g., t-Butoxycarbonyl (Boc). The amino side chain group of lysine isprotected as t-butoxycarbonyl, benzyloxycarbonyl orp-chlorobenzyloxycarbonyl (Z or Cl-Z). Acetamidomethyl, trityl, t-butyl,S-t-butyl or para-methylbenzyl (p-MBz) protection is used for cysteines.Hydroxy groups are protected as butyl or benzyl ethers and carboxylgroups are protected as butyl, benzyl (Bz) or cyclohexyl esters.

[0038] The peptides can be synthesized either from the C-terminus or theN-terminus, preferably the former. Prior to coupling the alpha-carboxylgroup (of a suitable protected amino acid) is activated. One skilled inthe art can activate the protected group in a number of ways. Forexample, one may use N,N′ dicyclohexylcarbodiimide (DCC),2(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate(HBTU), p-nitrophenyl esters (pNp), hydroxybenzotriazole ester (HOBt),N-hydroxy succinimidyl ester (OSu) mixed anhydride or symmetricalanhydride.

[0039] Solution synthesis of peptides is accomplished using conventionalmethods to form amide bonds. Typically, a protected Boc-amino acid whichhas a free carboxyl group is coupled to a protected amino acid which hasa free amino group using a suitable carbodiimide coupling agent, such asN,N′ dicyclohexyl carbodiimide (DCC), optionally in the presence of1-hydroxybenzotriazole (HOBT) and dimethylamino pyridine (DMAP).

[0040] In solution phase synthesis, the coupling reactions arepreferably carried out at low temperature (e.g., −20° C.) in suchsolvents as dichloromethane (DCM), dimethyl formamide (DMF), N-methylpyrrolidone (NMP), tetrahydrofuran (THF) acetonitrile (ACN) or dioxane.

[0041] If solid phase methods are used, the peptide is built upsequentially starting from the carboxy terminus and working towards theamino terminus of the peptide. Solid phase synthesis begins bycovalently attaching the C terminus of a protected amino acid to asuitable resin, such as4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin (Rink amideresin, H. Rink, Tetrahedron Letters 28, 3787, (1987)), 4-benzyloxybenzylalcohol resin (Wang resin, S. S. Wang, JACS, 95, 1328, (1973)) or4-hydroxymethyl phenoxy acetic acid resin.

[0042] In the solid phase synthesis, the first amino acid residue isnormally attached to an insoluble polymer. For example, two commonlyused polymers are polystyrene (1% cross-linked with divinyl benzene) and1% cross-linked polyacrylamide. These polymers are functionalised tocontain a reactive group, e.g., —OH, —NH₂ and —CH₂CI to link the firstamino acid of the targeted peptide (i.e., carboxy terminus). The choiceof the linkage between the first amino acid and the polymer is dictatedby the carboxy terminus of the peptide. For example, peptides having acarboxyl group at the C-terminus would be linked by an ester linkage andfor peptides with a carboxamide ending would have an amide linkage.

[0043] Once the first protected amino acid has been coupled to thedesired resin, the a amino protecting group is removed by treatment witha secondary amine such as piperidine, and the free carboxyl of the next(protected) amino acid is coupled to this amino group. This process iscarried out sequentially, without isolation of the intermediate, untilthe peptide of interest has been formed. The completed peptide may thenbe deblocked and/or cleaved from the resin in any order.

[0044] Preferred solvents for the coupling reactions include, but arenot limited to, dichloromethane (DCM), dimethyl formamide (DMF) andN-methyl pyrrolidone (NMP). After the desired sequence is synthesised,the peptide is deprotected and cleaved from the resin usingtrifluoroacetic acid or trifluoromethane sulphonic acid.

[0045] The preferred method for cleaving a peptide from the supportresin is to treat the resin supported peptide with trifluoroacetic acidin the presence of suitable cation and carbonium ion scavengers such asphenol, anisole, thioanisole, ethane dithiol, water or ethylmethylsulphide.

[0046] To obtain the compounds of the present invention, the syntheticpeptides may be cyclized/coupled using methods well known in the art.

[0047] For example coupling via a disulphide bond of two linear peptidesboth containing cysteine residues may be achieved in a selective mannerby reaction of the free thiol on one chain with a suitably activatedcysteine derivative on the other chain. A group which is especiallyuseful as a displaceable protecting group is theS-(carbomethoxy-sulphenyl) derivative. Examplary of this method is theprotection of both linear peptides' cysteine residues with theacetamidomethyl (Acm) group. Treatment of one chain with mercury (II)acetate followed by beta mercaptoethanol removes the acetamidomethylprotecting group. Treatment of the second chain withcarbomethoxysulphenyl chloride gives the activated species. Stirring ofthe two peptides in dilute aqueous solution at a pH of about 7 to 8causes displacement of the carbomethoxysulphenyl group and formation ofthe interchain disulphide.

[0048] If an intramolecular disulphide is to be formed then thecorresponding linear peptide can be completely deprotected and producedas a dimercaptan. Any oxidizing agent known in the art to be capable ofconverting a dimercaptan to a disulphide may then be used. Examplary ofsuch agents are an alkali metal ferricyanide, (e.g., potassium or sodiumferricyanide), oxygen gas, diiodomethane or iodine. The reaction isconducted in a suitable inert solvent, such as aqueous methanol orwater, at temperatures from about 0 to 40° C., under high dilution. ThepH is usually maintained at about 7 to 8. Cyclisation may be performedupon the peptide while it is still attached to the support resin orwhile other functional groups are still protected, but it is preferablyperformed on the deprotected free peptide.

[0049] In cases where two disulphides are to be formed between twolinear peptides, two types of cysteine thiol protecting groups can beemployed eg Acm and trityl. Each peptide would contain one of each typearranged so that one pair of cysteines to be coupled are protected withtrityl groups and the other pair with Acm. Independent removal of thetrityl group from each peptide would give two separate monothiolderivatives which can be coupled by activating the thiol on one peptidewith 2,2′dipyridyldisulphide and then adding the other monothiol peptideto give the bis(S-acetamido-methyl)disulphide-linked peptide. The seconddisulphide can be obtained by direct iodine oxidation of this product asdescribed by Kamber (B. Kamber, Helv. Chim. Acta 54, 927, (1971)), andKamber et. al. (B. Kamber et. al., Helv. Chim. Acta 63, 899, (1980)).

[0050] Peptide chains can also be coupled using a linking group such as—NH(CH₂)_(n)CO—. This is most easily achieved by employing theN^(a)-Fmoc derivative of the corresponding amino acid (NH₂(CH₂)_(n)COOH)and incorporating it into the growing peptide chain during conventionalsolid phase synthesis. A similar strategy can be employed to couplepeptide chains using the side chain carboxyl of an acidic amino acidsuch as glutamic acid, and the side chain amino of a basic amino acidsuch as lysine. In this case compounds such as the N⁶-g glutamyllysinederivative below may be incorporated into the growing peptide chainduring conventional solid phase synthesis

[0051] Coupling to the growing peptide chain is through the a carboxylof the glutamic acid residue and removal of the Fmoc grouping on thelysine a amino group provides a starting point for addition of furtheramino acids.

[0052] Alternatively the N^(a)-trityl protecting group may be employedon the glutamic acid residue and after coupling this may be removed with80% acetic acid and N-acetylated with acetic anhydride. Furthercouplings may proceed as previously described.

[0053] N-terminal N-acetyl groups may be introduced by acetylation ofthe free amino proteinated by removal of the amino protecting group,with acetic anhydride. C-terminal carboxamide groups are obtained byusing an appropriate solid phase synthesis resin such as the Rink amideresin.

[0054] As stated the peptides of the invention may also be preparedusing recombinant DNA techniques by expression of DNA encoding thepolypeptide sequence.

[0055] Accordingly, the invention extends to a recombinant peptide or afunctional derivative, analogue or variant thereof, which modulates bodyweight, substantially be means of modulating energy utilisation.

[0056] Any of the peptides mentioned herein form part of the inventionas recombinant peptides.

[0057] In a further aspect, the invention provides a process forpreparing a compound according to the invention which process comprisesexpressing DNA encoding said compound in a recombinant host cell andrecovering the product.

[0058] The DNA polymer comprising a nucleotide sequence that encodes thecompound also forms part of the invention.

[0059] The process of the invention may be performed by conventionalrecombinant techniques such as described in Maniatis et. al., MolecularCloning—A Laboratory Manual; Cold Spring Harbor, 1982 and DNA Cloningvols I, II and III (D. M. Glover ed., IRL Press Ltd).

[0060] In particular, the process may comprise the steps of:

[0061] i) preparing a replicable expression vector capable, in a hostcell, of expressing a DNA polymer comprising a nucleotide sequence thatencodes said compound;

[0062] ii) transforming a host cell with said vector;

[0063] iii) culturing said transformed host cell under conditionspermitting expression of said DNA polymer to produce said compound; and

[0064] iv) recovering said compound.

[0065] The invention also provides a process for preparing the DNApolymer by the condensation of appropriate mono-, di- or oligomericnucleotide units.

[0066] The preparation may be carried out chemically, enzymatically, orby a combination of the two methods, in vitro or in vivo as appropriate.Thus, the DNA polymer may be prepared by the enzymatic of appropriateDNA fragments, by conventional methods such as those described by D. M.Roberts et al in Biochemistry 1985, 24, 5090-5098.

[0067] The DNA fragments may be obtained by digestion of DNA containingthe required sequences of nucleotides with appropriate restrictionenzymes, by chemical synthesis, by enzymatic polymerisation on DNA orRNA templates, or by a combination of these methods. Preferably totalsynthesis of DNA fragments would be employed.

[0068] Digestion with restriction enzymes may be performed in anappropriate buffer at a temperature of 20°-70° C., preferably in avolume of 50 ml or less with 0.1-10 mg DNA.

[0069] Enzymatic polymerisation of DNA may be carried out in vitro usinga DNA polymerase such as DNA polymerase I (Klenow fragment) in anappropriate buffer containing the nucleoside triphosphates dATP, dCTP,dGTP and dTTP as required at a temperature of 10°-37° C. proteinrally ina volume of 50 ml or less.

[0070] Enzymatic ligation of DNA fragments may be carried out using aDNA ligase such as T4 DNA ligase in an appropriate buffer at atemperature of 4° C. to ambient, in a volume of 50 ml or less.

[0071] The chemical synthesis of the DNA polymer or fragments may becarried out by conventional phosphotriester, phosphite orphosphoramidite chemistry, using solid phase techniques such as thosedescribed in ‘Chemical and Enzymatic Synthesis of Protein Fragments—ALaboratory Manual’ (ed. H. G. Gassen and A. Lang), Verlag Chemie,Weinheim (1982), or in other scientific publications, for example M. J.Gait, H. W. D. Matthes, M. Singh, B. S. Sproat, and R. C. Titmas,Nucleic Acids Research, 1982, 10, 6243; B. S. Sproat and W V. Bannwarth,Tetrahedron Letters. 1983, 24, 5771, M. D. Matteucci and M. H Caruthers,Tetrahedron Letters, 1980, 21, 719; M. D. Matteucci and M. H. Caruthers,Journal of the American Chemical Society, 1981, 103, 3185; S. P. Adamset al., Journal of the American Chemical Society, 1983, 105, 661; N. D.Sinha, J. Biernat, J. McMannus, and H. Koester, Nucleic Acids Research,1984, 12, 4539; and H. W. D. Matthes et al., EMBO Journal, 1984, 3, 801.Preferably an automated DNA synthesizer is employed.

[0072] The DNA polymer is preferably prepared by ligating two or moreDNA molecules which together comprise a DNA sequence encoding thecompound.

[0073] The DNA molecules may be obtained by the digestion with suitablerestriction enzymes of vectors carrying the required coding sequences.

[0074] The precise structure of the DNA molecules and the way in whichthey are obtained depends upon the structure of the desired product. Thedesign of a suitable strategy for the construction of the DNA moleculecoding for the compound is a routine matter for the skilled worker inthe art.

[0075] The expression of the DNA polymer encoding the compound in arecombinant host cell may be carried out by means of a replicableexpression vector capable, in the host cell, of expressing the DNApolymer. The expression vector is novel and also forms part of theinvention.

[0076] The replicable expression vector may be prepared in accordancewith the invention, by cleaving a vector compatible with the host cellto provide a linear DNA segment having an intact replicon, and combiningsaid linear segment with one or more DNA molecules which, together withsaid linear segment, encode the compound, under ligating conditions.

[0077] The ligation of the linear segment and more than one DNA moleculemay be carried out simultaneously or sequentially as desired.

[0078] Thus, the DNA polymer may be preformed or formed during theconstruction of the vector, as desired.

[0079] The choice of vector will be determined in part by the host cell,which may be prokaryotic, such as E. Coli, or eukaryotic, such as mouseC127, mouse myeloma, chinese hamster ovary, fungi e.g. filamentous fungior unicellular yeast or an insect cell such as Drosophila. The host cellmay also be in a transgenic animal. Suitable vectors include plasmids,bacteriophages, cosmids and recombinant viruses derived from, forexample, baculoviruses or vaccinia.

[0080] The preparation of the replicable expression vector may becarried out conventionally with appropriate enzymes for restriction,polymerisation and ligation of the DNA, by procedures described in, forexample, Maniatis et al, cited above. Polymerisation and ligation may beperformed as described above for the preparation of the DNA polymer.Digestion with restriction enzymes may be performed in an appropriatebuffer at a temperature of 20°-70° C., proteinrally in a volume of 50 mlor less with 0.1-10 mg DNA.

[0081] The recombinant host cell is prepared, in accordance with theinvention, by transforming a host cell with a replicable expressionvector of the invention under transforming conditions. Suitabletransforming conditions are conventional and are described in, forexample, Maniatis et al., cited above, or “DNA Cloning” Vol. II, D. M.Glover ed., IRL Press Ltd, 1985.

[0082] The choice of transforming conditions is determined by the hostcell. Thus, a bacterial host such as E. coli may be treated with asolution of CaCl₂ (Cohen et al, Proc. Nat. Acad. Sci., 1973, 69, 2110)or with a solution comprising a mixture of RbCl, MnCl₂, potassiumacetate and glycerol, and then with 3-[N-morpholino]-propane-sulphonicacid, RbCl and glycerol. Mammalian cells in culture may be transformedby calcium co-precipitation of the vector DNA onto the cells.

[0083] The invention also extends to a vector comprising a compound ofthe invention.

[0084] The invention also extends to a host cell transformed with areplicable expression vector of the invention.

[0085] Culturing the transformed host cell under conditions permittingexpression of the DNA polymer is carried out conventionally, asdescribed in, for example, Maniatis et al and “DNA Cloning” cited above.Thus, preferably the cell is supplied with nutrient and cultured at atemperature below 45° C.

[0086] The expression product is recovered by conventional methodsaccording to the host cell. Thus, where the host cell is bacterial, suchas E. coli it may be lysed physically, chemically or enzymatically andthe protein product isolated from the resulting lysate. If the productis to be secreted from the bacterial cell it may be recovered from theperiplasmic space or the nutrient medium. Where the host cell ismammalian, the product may proteinrally be isolated from the nutrientmedium.

[0087] The DNA polymer may be assembled into vectors designed forisolation of stable transformed mammalian cell lines expressing theproduct; e.g. bovine papillomavirus vectors or amplified vectors inchinese hamster ovary cells (DNA cloning Vol. II D. M. Glover ed. IRLPress 1985; Kaufman, R. J. et al., Molecular and Cellular Biology 5,1750-1759, 1985; Pavlakis G. N. and Hamer, D. H., Proceedings of theNational Academy of Sciences (USA) 80, 397-401, 1983; Goeddel, D. V. etal., European Patent Application No. 0093619, 1983).

[0088] The peptides prepared by use of the above mentioned methods can,as required, be purified by a number of techniques. Preferredembodiments include gel filtration, chromatogaphy, reverse phase HPLCand crystallisation, especially chromatogaphy is used. The purifiedproducts can then be analysed for purity using HPLC, amino acidanalysis, amino acid sequencing and fast atom bombardment and/orelectrospray mass spectrometry.

[0089] The functional derivatives, analogues and variants of theproteins mentioned herein may be prepared by using conventional methodsanalogous to those mentioned herein.

[0090] As stated, the compounds of the invention are indicated to haveuseful pharmaceutical properties. Accordingly, there is also provided acompound of the invention for use as an active therapeutic substance.

[0091] In particular the compounds of the invention are considered to becapable of modulating body weight substantially by means of enhancingenergy utilization and are therefore of potential use in the treatmentof nutritional and metabolic disorders, particularly obesity anddiabetes.

[0092] The invention also provides a method for the treatment ofnutritional and metabolic disorders, which method comprises theadministration of an effective, pharmaceutically acceptable andnon-toxic amount of a compound of the invention.

[0093] The invention therefore further provides a pharmaceuticalcomposition comprising a compound of the invention and apharmaceutically acceptable carrier.

[0094] In use the active compound will normally be employed in the formof a pharmaceutical composition in association with a human orveterinary pharmaceutical carrier, diluent and/or excipient, althoughthe exact form of the composition will depend on the mode ofadministration. The active compound may, for example, be employed in theform of tablets, capsules, lozenges or syrups for oral administration;in the form of snuff, aerosol or nebulisable solution for inhalation; inthe form of sterile solutions for parenteral administration, or in theform of creams, lotions, liniments, gels, ointments or sprays fortopical administration. Parenteral routes of administration includeintravenous, intramuscular, subcutaneous, transcutaneous andintraperitoneal administration.

[0095] Also included are formulations of the above derivatives suitablefor use in subcutaneously implanted pumps or controlled release devices,in transdermal patches and as micronised powders suitable for intranasaladministration.

[0096] The dosage ranges for administration of the compounds of thepresent invention are those to produce the desired effect on thecondition to be treated, the dosage will proteinrally vary with age,extent or severity of the medical condition and contraindications, ifany. The dosage can vary from 0.001 mg/kg/day to 50 mg/kg/day, butpreferably 0.01 to 1.0 mg/kg/day.

[0097] Solid oral dosage forms may contain conventional excipients suchas diluents, for example lactose, microcrystalline cellulose, dicalciumphosphate, mannitol, magnesium carbonate, glycine, dextrose, sucrose,starch, mannitol, sorbitol and calcium carbonate; binders, for exampleliquid glucose, syrup, acacia, gelatin, starch mucilage,methylcellulose, polyvinylpyrrolidone, alginates, and pregelatinisedstarch; disintegrants for example starch, alginic acid, microcrystallinecellulose, pectin, cross-linked polyvinylpyrrolidone, sodium starchglycollate and sodium carboxymethyl-cellulose; glidants for example talcand silica; lubricants for example stearic acid and magnesium stearate;preservatives for example sorbic acid and methyl or propylparahydroxybenzoate, or pharmaceutically acceptable wetting agents forexample sodium lauryl sulphate.

[0098] Capsules consist of a shell, normally of gelatin together withother ingredients for example, glycerol, sorbitol, surface-activeagents, opaque fillers, preservatives, sweeteners, flavours and colours.The contents of capsules may include diluents, lubricants anddisintegrants. Tablets consist of compressed powders or granules, may becoated or uncoated and may be designed so as to dissolve, disperse oreffervesce before administration to the patient, or to dissolve ordisperse in the gastrointestinal tract either immediately afterswallowing, or, for example in the case of tablets with acid-insolublecoatings, at later times. Tablets usually contain excipients such asdiluents, binders, disintegrants, glidants, lubricants and may containcolours and flavours. Effervescent tablets proteinrally contain acidstogether with carbonates or bicarbonates. Coatings for tablets mayconsist of natural or synthetic resins, gums, insoluble fillers, sugars,plasticisers, polyhydric alcohols and waxes and may also contain coloursand flavours. Lozenges and pastilles are intended to dissolve in themouth. Lozenges may be moulded or compressed and usually have aflavoured base. Pastilles are moulded from a base of gelatin andglycerol or acacia and sucrose. They may contain a preservative as wellas colours and flavours.

[0099] Film-coating resins include cellulose derivatives, zein, vinylpolymers and acrylic resins, and coating compositions usually includeplasticisers, such as castor oil or glycerol triacetate. Enteric-coatingresins include cellulose acetate phthalate and copolymers of methacrylicacid.

[0100] Solid compositions suitable for oral administration may beobtained by conventional methods of blending, filling, granulation,tabletting or the like. Repeated blending operations may be used todistribute the active agent throughout those compositions employinglarge quantities of fillers.

[0101] Liquid compositions suitable for oral administration may be inthe form of, for example, elixirs, mixtures, concentrated solutions,suspensions, emulsions or linctuses. They may be presented as a dryproduct for reconstitution with water or other suitable vehicle beforeuse. Such liquid compositions may contain conventional excipients suchas suspending agents, for example sucrose, sorbitol, gelatin, methylcellulose, carboxymethylcellulose, hydroxypropyl methyl cellulose,sodium alginate, Xanthan gum, acacia, carageenan, silica, aluminiumstearate gel; emulsifying agents, for example lecithin, acacia, sorbitanmono-oleate; aqueous or non-aqueous vehicles which include edible oils,oily esters, for example esters of glycerol, ethanol, glycerol;buffering agents for example citrates and phosphates of alkali metals;preservatives, for example sodium benzoate, sorbic acid, methyl orpropyl parahydroxybenzoate; and if desired, conventional flavouring andcolouring agents.

[0102] The composition may be implanted subcutaneously, for example inthe form of a compressed tablet or slow release capsule.

[0103] Alternatively, compositions suitable for injection may be in theform of solutions, suspensions or emulsions, or dry powders which aredissolved or suspended in a suitable vehicle prior to use.

[0104] Fluid unit dosage forms are prepared utilising the compound and apyrogen-free sterile vehicle. The compound, depending on the vehicle andconcentration used, can be either dissolved or suspended in the vehicle.Solutions may be used for all forms of parenteral administration, andare particularly used for intravenous infection. In preparing solutionsthe compound can be dissolved in the vehicle, the solution being madeisotonic if necessary by addition of sodium chloride and sterilised byfiltration through a sterile filter using aseptic techniques beforefilling into suitable sterile vials or ampoules and sealing.Alternatively, if solution stability is adequate, the solution in itssealed containers may be sterilised by autoclaving. Advantageouslyadditives such as buffering, solubilising, stabilising, preservative orbactericidal, suspending or emulsifying agents and/or local anaestheticagents may be dissolved in the vehicle.

[0105] Dry powders which are dissolved or suspended in a suitablevehicle prior to use may be prepared by filling pre-sterilised drugsubstance and other ingredients into a sterile container using aseptictechnique in a sterile area. Alternatively the drug and otheringredients may be dissolved in an aqueous vehicle, the solution issterilised by filtration and distributed into suitable containers usingaseptic technique in a sterile area. The product is then freeze driedand the containers are sealed aseptically.

[0106] Parenteral suspensions, suitable for intramuscular, subcutaneousor intradermal injection, are prepared in substantially the same manner,except that the sterile compound is suspended in the sterile vehicle,instead of being dissolved and sterilisation cannot be accomplished byfiltration. The compound may be isolated in a sterile state oralternatively it may be sterilised after isolation, e.g. by gammairradiation. Advantageously, a suspending agent for examplepolyvinylpyrrolidone is included in the composition to facilitateuniform distribution of the compound.

[0107] In a further aspect there is provided a method of treatingnutritional and metabolic disorders, which comprises administering tothe sufferer an effective, non-toxic amount of a compound of theinvention.

[0108] The invention also provides the use of a compound of theinvention for the manufacture of a medicament for treating nutritionaland metabolic disorders, such as obesity and diabetes.

[0109] No unexpected toxicological effects are expected when compoundsof the invention are administered in accordance with the presentinvention.

[0110] The following examples illustrate compounds of the invention.

[0111] Pharmacological Methods: The activity of the compounds of theinvention are assessed according to the methodology set our below:

EFFECT OF LEPTIN FRAGMENTS ON FOOD INTAKE IN SD RATS

[0112] Surgery

[0113] Rats are pre-treated with Synulox (0.1 ml/100 g) approx 1 hourbefore anaesthesia, and then anaesthetised with Domitor (0.04 ml/100 gi.m.) and sublimase (0.9 ml/100 g i.p.) Each rat has a cannula implantedstereotaxically into the lateral brain ventricle under sterileconditions. Anaesthesia is then reversed using Antisedan and Nubain (50%v/v:50% v/v 0.02 ml/100 g) I.P. After surgery each rat receives 0.05 mlZenecarp.

[0114] Experimental Procedure

[0115] Experiment 1: Following surgery the body weight of each animalwas monitored daily throughout the procedure.

[0116] In order to verify that the cannula was in the lateral ventricle,Angiotensin II (100 ng/5 μl) was injected icv and water intake wasmonitored for 5 min after injection.

[0117] 24 hour food intake was measured on day 5 and 6 after surgery. Onday 6 the animals were divided according to their body weight into 3groups (a, b and c, 8 rats per group) and then fasted overnight. On theday of experiment (day 7) rats were injected icv as follows::

[0118] group a-vehicle (PBS, phosphate buffer solution, 5 μl/rat);

[0119] group b-human leptin (11.5 μg/5 μl); and

[0120] group c-leptin tryptic digest (30 μg/5 μl).

[0121] A known quantity of food in excess of the daily requirement wassupplied to the rats immediately after the icv injection procedure wascompleted. Food intake and body weight were then measured 24 h later.The results obtained are shown in Table 1.

[0122] Experiment 2: A separate group of animals was prepared exactly asdescribed above but on the day of experiment after overnight fast,groups of rats were injected as follows:

[0123] group a-vehicle (PBS 5 μl/rat);

[0124] group b-human leptin (8.75 μg/5 μl);

[0125] group c-murine leptin (10 μg/5 μl);

[0126] group d-ob 57-74, sequence VTGLDFIPGLHPILTLSK (3.33 μg/5 μl);

[0127] Again a pre-weight quantity of food, in excess of the dailyrequirement, was re-given following the injection; change in body weightand the quantity of food consumed were recorded 24 h later. The resultsobtained are shown in Table 2.

[0128] Results TABLE 1 Effect of human leptin and its fragments givenintracerebroventricularly on body weight and food intake in SD rats. 24h food intake Bwt Change (g) (g/24 h) Vehicle (5 μl/rat, n = 8) 36.68 ±2.5  31.44 ± 1.2  h-Leptin 30.62 ± 1.5* 22.88 ± 2.05* (11.5 μg/rat, n =8) h-Leptin tryptic  34.6 ± 1.99 23.88 ± 1.2*  digest (30 μg/rat, n = 8)

[0129] TABLE 2 Effect of human leptin (h-leptin), murine leptin(m-leptin) and leptin fragment 57-74 (VTGLDFIPGLHPILTLSK) givenintracerebroventricularly on body weight and food intake in SD rats. 24h food intake Bwt Change (g) (g/24 h) Vehicle (5 μl/rat, n = 8) 32.16 ±0.8 30.55 ± 0.67 h-Leptin  33.7 ± 1.6 24.5 ± 3.7 (8.75 μg/rat, n = 8)m-Leptin  31.3 ± 1.7  23.7 ± 2.18* (10 μg/rat, n = 8) Leptin fragment 33.6 ± 0.9  26.8 ± 1.2* 57-74 (3.33 μg/rat, n = 8)

1. A peptide or a functional derivative, analogue or variant thereof,which modulates body weight, substantially be means of modulating energyutilisation.
 2. A peptide or a functional derivative, analogue orvariant thereof, which reduces body weight, substantially be means ofmodulating energy utilisation.
 3. A peptide according to claim 1 orclaim 2, wherein the peptide is a fragment of an ob protein, or afunctional derivative, analogue or variant thereof.
 4. A peptideaccording to any one of claims 1 to 3, selected from a fragment of humanob protein in the list: ob21-26 , ob27 -32 , ob33 -36 , ob37-41 , ob42-54, ob55 -56, ob57-74, ob93 -105, ob106-115, ob116-149 and ob150-167.5. A peptide according to any one of claims 1 to 4, wherein the peptidehas the amino acid sequence VTGLDFIPGLHPILTLSK.
 6. A peptide accordingto claim 1, formed from one or more of the peptides of claim
 4. 7. Apeptide according to claim 1, formed from two contiguous members of thepeptides of claim
 4. 8. A synthetic or recombinant peptide, being apeptide according to any one of claims 1 to
 7. 9. A nucleotide sequencethat encodes a peptide of any one of claims 1 to
 7. 10. A vectorcomprising a nucleotide sequence that encodes a peptide of any one ofclaims 1 to
 7. 11. A host cell transformed with a replicable expressionvector of claim
 9. 12. A process for the preparation of a peptideaccording to claim 1, or a functional derivative thereof, the processcomprising the steps of: hydrolysing the peptide into at least twopeptide fragments; separating the peptide fragments; and optionallythereafter preparing a functional derivative thereof.
 13. A process forpreparing a peptide according to any one of claims 1 to 7, which processcomprises expressing DNA encoding said peptide in a recombinant hostcell and recovering the product.
 14. A process accoring to claim 13,comprising the steps of: i) preparing a replicable expression vectorcapable, in a host cell, of expressing a DNA polymer comprising anucleotide sequence that encodes the required peptide; ii) transforminga host cell with said vector; iii) culturing said transformed host cellunder conditions permitting expression of said DNA polymer to producesaid peptide; and iv) recovering said peptide.
 15. A pharmaceuticalcomposition comprising a compound according to claim 1, and apharmaceutically acceptable carrier.
 16. A compound according to claim1, for use as an active therapeutic substance.
 17. A compound accordingto claim 15, for use in the treatment of nutritional and metabolicdisorders.
 18. A method for the treatment of nutritional and metabolicdisorders, which method comprises the administration of an effective,pharmaceutically acceptable and non-toxic amount of a compound accordingto claim
 1. 19 The use of a compound according to claim 1, for themanufacture of a medicament for treating nutritional and metabolicdisorders.